Leveraging gains from African Center for Integrated Laboratory Training to combat HIV epidemic in sub-Saharan Africa.

BACKGROUND: In sub-Saharan Africa, there is dearth of trained laboratorians and strengthened laboratory systems to provide adequate and quality laboratory services for enhanced HIV control. In response to this challenge, in 2007, the African Centre for Integrated Laboratory Training (ACILT) was established in South Africa with a mission to train staffs from countries with high burdens of diseases in skills needed to strengthen sustainable laboratory systems. This study was undertaken to assess the transference of newly gained knowledge and skills to other laboratory staff, and to identify enabling and obstructive factors to their implementation. METHODS: We used Kirkpatrick model to determine training effectiveness by assessing the transference of newly gained knowledge and skills to participant's work environment, along with measuring enabling and obstructive factors. In addition to regular course evaluations at ACILT (pre and post training), in 2015 we sent e-questionnaires to 867 participants in 43 countries for course participation between 2008 and 2014. Diagnostics courses included Viral Load, and systems strengthening included strategic planning and Biosafety and Biosecurity. SAS v9.44 and Excel were used to analyze retrospective de-identified data collected at six months pre and post-training. RESULTS: Of the 867 participants, 203 (23.4%) responded and reported average improvements in accuracy and timeliness in Viral Load programs and to systems strengthening. For Viral Load testing, frequency of corrective action for unsatisfactory proficiency scores improved from 57 to 91%, testing error rates reduced from 12.9% to 4.9%; 88% responders contributed to the first national strategic plan development and 91% developed strategies to mitigate biosafety risks in their institutions. Key enabling factors were team and management support, and key obstructive factors included insufficient resources and staff's resistance to change. CONCLUSIONS: Training at ACILT had a documented positive impact on strengthening the laboratory capacity and laboratory workforce and substantial cost savings. ACILT's investment produced a multiplier effect whereby national laboratory systems, personnel and leadership reaped training benefits. This laboratory training centre with a global clientele contributed to improve existing laboratory services, systems and networks for the HIV epidemic and is now being leveraged for COVID-19 testing that has infected 41,332,899 people globally.

Diagnosis of Human Immunodeficiency Virus Infection.

HIV diagnostics have played a central role in the remarkable progress in identifying, staging, initiating, and monitoring infected individuals on life-saving antiretroviral therapy. They are also useful in surveillance and outbreak responses, allowing for assessment of disease burden and identification of vulnerable populations and transmission "hot spots," thus enabling planning, appropriate interventions, and allocation of appropriate funding. HIV diagnostics are critical in achieving epidemic control and require a hybrid of conventional laboratory-based diagnostic tests and new technologies, including point-of-care (POC) testing, to expand coverage, increase access, and positively impact patient management. In this review, we provide (i) a historical perspective on the evolution of HIV diagnostics (serologic and molecular) and their interplay with WHO normative guidelines, (ii) a description of the role of conventional and POC testing within the tiered laboratory diagnostic network, (iii) information on the evaluations and selection of appropriate diagnostics, (iv) a description of the quality management systems needed to ensure reliability of testing, and (v) strategies to increase access while reducing the time to return results to patients. Maintaining the central role of HIV diagnostics in programs requires periodic monitoring and optimization with quality assurance in order to inform adjustments or alignment to achieve epidemic control.

Long-term immunological responses to treatment among HIV-2 patients in Côte d'Ivoire.

BACKGROUND: Studies indicate that responses to HIV-2 treatment regimens are worse than responses to HIV-1 regimens during the first 12 months of treatment, but longer-term treatment responses are poorly described. We utilized data from Côte d'Ivoire's RETRO-CI laboratory to examine long-term responses to HIV-2 treatment. METHODS: Adult (≥15 years) patients with baseline CD4 counts < 500 cells/µl that initiated treatment at one of two HIV treatment centers in Abidjan, Côte d'Ivoire between 1998 and 2004 were included in this retrospective cohort study. Patients were stratified by baseline CD4 counts and survival analyses were employed to examine the relationship between HIV type and time to achieving CD4 ≥ 500 cells/µl during follow up. RESULTS: Among 3487 patients, median follow-up time was 4 years and 57% had documented ART regimens for > 75% of their recorded visits. Kaplan-Meier estimates for achievement of CD4 ≥ 500 cells/µl after 6 years of follow-up for patients in the lower CD4 strata (< 200 cells/µl) were 40% (HIV-1), 31% (HIV-dual), and 17% (HIV-2) (log-rank p < 0.001). Cox Regression indicated that HIV-1 was significantly associated with achievement of CD4 ≥ 500 cells/µl during follow-up, compared to HIV-2. CONCLUSIONS: Sub-optimal responses to long-term HIV-2 treatment underscore the need for more research into improved and/or new treatment options for patients with HIV-2. In many West African countries, effective treatment of both HIV-1 and HIV-2 will be essential in the effort to reach epidemic control.

Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

Point of Care CD4 Testing in National Household Surveys - Results and Quality Indicators from Eleven Population-Based HIV Impact Assessment (PHIA) Surveys.

Population-based HIV Impact Assessments (PHIAs) are national household (HH) surveys that provide HIV diagnosis and CD4 testing with an immediate return of results. Accurate CD4 results improve HIV-positive participants' clinical care and inform the effectiveness of HIV programs. Here, we present CD4 results from the PHIA surveys that were conducted in 11 countries in sub-Saharan Africa between 2015 and 2018. All of the HIV-positive participants and 2 to 5% of the HIV-negative participants were offered Pima CD4 (Abbott, IL, USA) point-of-care (POC) tests. The quality of the CD4 test was ensured by conducting instrument verification, comprehensive training, quality control, a review of testing errors and an analysis of unweighted CD4 data by HIV status, age, gender, and antiretroviral (ARV) treatment status. Overall, CD4 testing was completed for 23,085 (99.5%) of the 23,209 HIV-positive and 7,329 (2.7%) of the 270,741 negative participants in 11 surveys. The instrument error rate was 11.3% (range, 4.4% to 15.7%). The median CD4 values among HIV-positive and HIV-negative participants (aged 15+) were 468 cells/mm3 (interquartile range [IQR], 307 to 654) and 811 cells/mm3 (IQR, 647 to 1,013), respectively. Among the HIV-positive participants (aged 15+), those with detectable ARVs had higher CD4 values (508 cells/mm3) than those with undetectable ARVs (385.5 cells/mm3). Among the HIV-positive participants (aged 15+), 11.4% (2,528/22,253) had a CD4 value of less than 200 cells/mm3, and approximately half of them (1,225/2,528 = 48.5%) had detectable ARVs, whereas 51.5% (1,303/2,528) had no detectable ARVs (P < 0.0001). We successfully implemented high quality POC CD4 testing using Pima instruments. Our data come from nationally representative surveys in 11 countries and provide unique insights regarding the CD4 distribution among HIV-positive individuals as well as the baseline CD4 values among HIV-negative individuals. IMPORTANCE The manuscript describes CD4 levels among HIV-positive individuals and baseline CD4 levels among HIV-negative individuals from 11 sub-Saharan countries, thereby highlighting the importance of CD4 markers in the context of the HIV epidemic. Despite increased ARV access in each country, advanced HIV disease (CD4 < 200 cells/mm3) persists among approximately 11% of HIV-positive individuals. Therefore, it is important that our findings are shared with the scientific community to assist with similar implementations of point-of-care testing and to conduct a review of HIV programmatic gaps.

Progress in scale up of HIV viral load testing in select sub-Saharan African countries 2016-2018.

INTRODUCTION: We assessed progress in HIV viral load (VL) scale up across seven sub-Saharan African (SSA) countries and discussed challenges and strategies for improving VL coverage among patients on anti-retroviral therapy (ART). METHODS: A retrospective review of VL testing was conducted in Côte d'Ivoire, Kenya, Lesotho, Malawi, Namibia, Tanzania, and Uganda from January 2016 through June 2018. Data were collected and included the cumulative number of ART patients, number of patients with ≥ 1 VL test result (within the preceding 12 months), the percent of VL test results indicating viral suppression, and the mean turnaround time for VL testing. RESULTS: Between 2016 and 2018, the proportion of PLHIV on ART in all 7 countries increased (range 5.7%-50.2%). During the same time period, the cumulative number of patients with one or more VL test increased from 22,996 to 917,980. Overall, viral suppression rates exceeded 85% for all countries except for Côte d'Ivoire at 78% by June 2018. Reported turnaround times for VL testing results improved in 5 out of 7 countries by between 5.4 days and 27.5 days. CONCLUSIONS: These data demonstrate that remarkable progress has been made in the scale-up of HIV VL testing in the seven SSA countries.

Dried blood spot specimens are a suitable alternative sample type for HIV-1 viral load measurement and drug resistance genotyping in patients receiving first-line antiretroviral therapy.

BACKGROUND: Antiretroviral therapy (ART) is being administered in developing nations at unprecedented numbers following the World Health Organization's (WHO) development of standardized first-line drug regimens. To ensure continued efficacy of these drug regimens, WHO recommends monitoring virological responses and development of human immunodeficiency virus (HIV) drug resistance (HIVDR) in HIV-infected patients in a prospective cohort. The current study compared dried fluid spot specimens with the reference standard plasma specimens as a practical tool for viral load (VL) and HIVDR genotyping in resource-limited settings. METHODS: Dried blood spot (DBS), dried plasma spot (DPS), and plasma specimens were collected from 173 -patients receiving ART at 2 hospital sites in Abuja, Nigeria. HIV-1 VL analysis was performed using NucliSENS EasyQ HIV-1 v1.1 RUO test kits. Genotyping of the HIV-1 pol gene was performed using a broadly sensitive in-house assay. RESULTS: Direct comparison of VL levels showed that DBS specimens, and not DPS specimens, gave results comparable to those of plasma specimens (P = .0619 and .0007, respectively); however, both DBS and DPS specimens had excellent correlation with plasma specimens in predicting virological failure (VL, ≥1000 copies/mL) in patients (κ = 0.78 and 0.83, respectively). Of the 18 specimens with a plasma VL ≥1000 copies/mL, HIVDR genotyping rates were 100% in DBS and 38.9% in DPS specimens, and DBS specimens identified 61 of 65 HIVDR mutations (93.8%) identified in plasma specimens. CONCLUSIONS: Our results indicate that DBS specimens could be used for surveys to monitor HIVDR prevention failure in resource-limited settings.

Virological response and HIV drug resistance 12 months after antiretroviral therapy initiation at 2 clinics in Nigeria.

This report describes a pilot study, conducted in Nigeria, of the World Health Organization protocol for monitoring human immunodeficiency virus (HIV) drug resistance (HIVDR) and associated program factors among patients receiving first-line antiretroviral therapy (ART). In 2008, 283 HIV-infected patients starting ART were consecutively enrolled at 2 ART clinics in Abuja. Twelve months after ART initiation, 62% were alive and on first-line ART, 3% had died, 1% had transferred out of the program, and 34% were lost to follow-up. Among patients on first-line ART at 12 months, 90% had viral suppression. However, in view of the high loss to follow-up rate (34%), strategies for patient retention and tracking are critical to minimize possible HIVDR and optimize treatment outcomes.

CD8 T Cells in old mice contribute to the innate immune response to Mycobacterium tuberculosis via interleukin-12p70-dependent and antigen-independent production of gamma interferon.

Elderly individuals have increased morbidity and mortality associated with infectious diseases due in part to the progressive age-associated decline in immune function. Despite this, the old mouse model of Mycobacterium tuberculosis infection has revealed a CD8- and gamma interferon (IFN-gamma)-dependent early resistance to infection. In this study, we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to M. tuberculosis. Following a low-dose aerosol infection with M. tuberculosis, CD8 T cells were identified as being a dominant source of IFN-gamma expression in the lungs of old mice early after infection, before the typical onset of antigen-specific immunity. In addition, M. tuberculosis-induced IFN-gamma production by CD8 T cells isolated from naïve old mice was major histocompatibility complex class I independent but was dependent on interleukin-12p70, confirming an innate role of CD8 T cells during M. tuberculosis infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-gamma levels in response to M. tuberculosis infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as being one possible mechanism by which CD8 T cells from old mice produce enhanced levels of IFN-gamma. Additional characterizations of the signaling events that lead to enhanced innate IFN-gamma production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.

Interleukin-12 is sufficient to promote antigen-independent interferon-gamma production by CD8 T cells in old mice.

Numerous functional defects have been identified in naive T cells from aged mice, including deficiencies in proliferation, cytokine production and signal transduction. It is well documented that the ratio of naïve to memory T cells significantly decreases with age resulting in the majority of T cells from aged hosts expressing activated/memory T-cell markers (CD44(hi)), yet it is unclear whether T cells with a CD44(hi) phenotype in aged hosts are functionally equivalent to T cells with a similar phenotype in young hosts. We have identified a population of CD44(hi) CD8 T cells in old mice that are capable of secreting interferon-gamma (IFN-gamma) in response to interleukin-12 (IL-12) stimulation. This occurred in the absence of T-cell receptor engagement, a function that was not observed in CD8 T cells from young mice. This phenotype was associated with increased IL-12 receptor beta2 gene expression and IL-12 induced signal transducer and activator of transcription 4 (STAT-4) activation, even when CD8 T-cell numbers from young and old mice were normalized for CD44(hi) expression. Furthermore, we demonstrate that IL-12-induced STAT-4 activation was required for T helper type 1 (Th1) cytokine-induced IFN-gamma production in CD8 T cells. These data illustrate that old mice possess a specialized subset of CD44(hi) CD8 T cells with an enhanced responsiveness to IL-12, enabling these cells to produce substantial amounts of IFN-gamma in response to Th1 cytokine stimulation. We have therefore identified a functional difference in the populations of CD44(hi) CD8 T cells from young and old mice, and believe that understanding age-associated immunological changes is essential for helping the elderly combat deadly diseases.

Field Validation of Limiting-Antigen Avidity Enzyme Immunoassay to Estimate HIV-1 Incidence in Cross-Sectional Survey in Swaziland.

Reliable and accurate laboratory assays to detect recent HIV-1 infection have potential as simple and practical methods of estimating HIV-1 incidence in cross-sectional surveys. This study describes validation of the limiting-antigen (LAg) avidity enzyme immunoassay (EIA) in a cross-sectional national survey, conducted in Swaziland, comparing it to prospective follow-up incidence. As part of the Swaziland HIV-1 Incidence Measurement Survey (SHIMS), 18,172 individuals underwent counseling and HIV rapid testing in a household-based, population survey conducted from December 2010 to June 2011. Plasma samples from HIV-positive persons were classified as recent infections using an incidence testing algorithm with LAg-Avidity EIA (normalized optical density ≤1.5) followed by viral load (VL ≥1,000 copies/mL). All HIV-seronegative samples were tested for acute HIV-1 infection by nucleic acid amplification test (NAAT) pooling. HIV-seronegative individuals who consented to follow-up were retested ∼6 months later to detect observed HIV-1 seroconversion. HIV-1 incidence estimates based on LAg+VL and NAAT were calculated using assay-specific parameters and were compared with prospective incidence estimate. A total of 5,803 (31.9%) of 18,172 survey participants tested HIV seropositive; of these 5,683 (97.9%) were further tested with LAg+VL algorithm. The weighted annualized incidence from the longitudinal cohort study was 2.4% (95% confidence interval 2.0-2.7). Based on cross-sectional testing of HIV positives with LAg+VL algorithm, overall weighted annualized HIV-1 incidence was 2.5% (2.0-3.0), whereas NAAT-based incidence was of 2.6%. In addition, LAg-based incidence in men (1.8%; 1.2-2.5) and women (3.2%; 2.4-3.9) were similar to estimates based on observed incidence (men = 1.7%, women = 3.1%). Changes in HIV-1 incidence with age in men and women further validate plausibility of the algorithm. These results demonstrate that the LAg EIA, in a serial algorithm with VL, is a cost-effective tool to estimate HIV-1 incidence in cross-sectional surveys.

Age dependent increase in early resistance of mice to Mycobacterium tuberculosis is associated with an increase in CD8 T cells that are capable of antigen independent IFN-gamma production.

The lungs of naïve 18-month-old mice contain an abundant resident population of CD8 T cells that express typical markers of memory, express elevated levels of Th1 cytokine receptors on their surface, and are capable of non-specific IFN-gamma production in response to a Th1 cytokine cocktail. In this study we characterize this population of CD8 T cells in the lungs and spleens of mice with increasing age. In general, the proportion of CD8 T cells expressing markers of memory and Th1 cytokine receptors increased with age. The enhanced ability of CD8 T cells to produce IFN-gamma in an antigen independent manner followed this pattern as well, beginning to increase between 6 and 12 months of age. Interestingly, the phenotypic and functional age-related changes in CD8 T cells were also associated with a progressive age-related increase in early resistance to Mycobacterium tuberculosis. Taken together, these data suggest that as mice age a population of memory CD8 T cells, that are capable of contributing to innate immune responses to M. tuberculosis, gradually emerges and could be relevant for developing strategies to enhance immunity in the elderly.

Viral Genetic Diversity and Polymorphisms in a Cohort of HIV-1-Infected Patients Eligible for Initiation of Antiretroviral Therapy in Abuja, Nigeria.

Studying the genetic diversity and natural polymorphisms of HIV-1 would benefit our understanding of HIV drug resistance (HIVDR) development and predict treatment outcomes. In this study, we have characterized the HIV-1 genetic diversity and natural polymorphisms at the 5' region of the pol gene encompassing the protease (PR) and reverse transcriptase (RT) from 271 plasma specimens collected in 2008 from HIV-1-infected patients who were eligible for initiating antiretroviral therapy in Abuja (Nigeria). The analysis indicated that the predominant subtype was subtype G (31.0%), followed by CRF02-AG (19.2 %), CRF43-02G (18.5%), and A/CRF36-cpx (11.4%); the remaining (19.9%) were other subtypes and circulating (CRF) and unique (URF) recombinant forms. Recombinant viruses (68.6%) were the major viral strains in the region. Eighty-four subtype G sequences were further mainly classified into two major and two minor clusters; sequences in the two major clusters were closely related to the HIV-1 strains in two of the three major subtype G clusters detected worldwide. Those in the two minor clusters appear to be new subtype G strains circulating only in Abuja. The pretreatment DR prevalence was <3%; however, numerous natural polymorphisms were present. Eleven polymorphic mutations (G16E, K20I, L23P, E35D, M36I, N37D/S/T, R57K, L63P, and V82I) were detected in the PR that were subtype or CRF specific while only three mutations (D123N, I135T, and I135V) were identified in the RT. Overall, this study indicates an evolving HIV-1 epidemic in Abuja with recombinant viruses becoming the dominant strains and the emergence of new subtype G strains; pretreatment HIVDR was low and the occurrence of natural polymorphism in the PR region was subtype or CRF dependent.

Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes.

BACKGROUND: Mean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus. METHODS: A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs. RESULTS: Using different statistical methods, MDRI values ranged from 88-94 days at cutoff ODn = 1.0 to 177-183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing for misclassification among long-term infections indicated that overall PFRs were 0.6% to 2.5% at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C). CONCLUSIONS: Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118-142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.

Evaluation of dried blood spots collected on filter papers from three manufacturers stored at ambient temperature for application in HIV-1 drug resistance monitoring.

As more HIV-infected people gain access to antiretroviral therapy (ART), monitoring HIV drug resistance (HIVDR) becomes essential to combat both acquired and transmitted HIVDR. Studies have demonstrated dried blood spots (DBS) are a suitable alternative in HIVDR monitoring using DBS collected on Whatman 903 (W-903). In this study, we sought to evaluate two other commercially available filter papers, Ahlstrom 226 (A-226) and Munktell TFN (M-TFN), for HIVDR genotyping following ambient temperature storage. DBS were prepared from remnant blood specimens collected from 334 ART patients and stored at ambient temperature for a median time of 30 days. HIV-1 viral load was determined using NucliSENS EasyQ® HIV-1 v2.0 RUO test kits prior to genotyping of the protease and reverse transcriptase regions of the HIV-1 pol gene using an in-house assay. Among the DBS tested, 26 specimens had a viral load ≥ 1000 copies/mL in all three types of filter paper and were included in the genotyping analysis. Genotyping efficiencies were similar between DBS collected on W-903 (92.3%), A-226 (88.5%), and M-TFN (92.3%) filter papers (P = 1.00). We identified 50 DR-associated mutations in DBS collected on W-903, 33 in DBS collected on A-226, and 48 in DBS collected on M-TFN, resulting in mutation detection sensitivities of 66.0% for A-226 and 88.0% for M-TFN when compared to W-903. Our data indicate that differences among filter papers may exist at this storage condition and warrant further studies evaluating filter paper type for HIVDR monitoring.

Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.

UNLABELLED: Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX. CONCLUSIONS: The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.

TLR-2 independent recognition of Mycobacterium tuberculosis by CD11c+ pulmonary cells from old mice.

The elderly are particularly susceptible to infectious diseases such as influenza, bacterial pneumonia, and tuberculosis. Current vaccines are only partially protective in old age, which makes the elderly a critical target group for the development of new vaccine strategies. The recognition of pathogens via toll like receptors (TLR) and the subsequent generation of pro-inflammatory cytokines has generated interest in incorporating TLR agonists into new vaccines to enhance immunogenicity. However, TLR function is reportedly decreased in old age, leading to questions regarding the benefit of including TLR agonists into vaccines for the elderly. It is critical that we understand the function and role of TLRs in aged hosts prior to approving new TLR based adjuvants for vaccines that will be delivered to the elderly. In this study we determine the ability of TLRs on pulmonary macrophages from old mice to recognize and respond to infection with the virulent pathogen Mycobacterium tuberculosis (M. tb). Although pulmonary (CD11c(+)) cells from old mice were fully capable of producing cytokines in response to M. tb infection, we demonstrate that in contrast to young mice, M. tb induced cytokine production occurred independently of TLR-2. Our data indicate that the inclusion of TLR-2 agonists into new vaccines may not be fully effective in the elderly population. Investigation into such age-related differences in TLR function is of critical importance for the design of effective vaccines that will protect the elderly against infectious diseases.

CCL5 participates in early protection against Mycobacterium tuberculosis.

Control of M.tb, the causative agent of TB, requires immune cell recruitment to form lung granulomas. The chemokines and chemokine receptors that promote cell migration for granuloma formation, however, are not defined completely. As immunity to M.tb manifests slowly in the lungs, a better understanding of specific roles for chemokines, in particular those that promote M.tb-protective T(H)1 responses, may identify targets that could accelerate granuloma formation. The chemokine CCL5 has been detected in patients with TB and implicated in control of M.tb infection. To define a role for CCL5 in vivo during M.tb infection, CCL5 KO mice were infected with a low dose of aerosolized M.tb. During early M.tb infection, CCL5 KO mice localized fewer APCs and chemokine receptor-positive T cells to the lungs and had microscopic evidence of altered cell trafficking to M.tb granulomas. Early acquired immunity and granuloma function were transiently impaired when CCL5 was absent, evident by delayed IFN-gamma responses and poor control of M.tb growth. Lung cells from M.tb-infected CCL5 KO mice eventually reached or exceeded the levels of WT mice, likely as a result of partial compensation by the CCL5-related ligand, CCL4, and not because of CCL3. Finally, our results suggest that most T cells use CCR5 but not CCR1 to interact with these ligands. Overall, these results contribute to a model of M.tb granuloma formation dependent on temporal regulation of chemokines rather than on redundant or promiscuous interactions.